Carol J. Cogswell

Linear phase imaging using differential interference contrast microscopy.

We propose an extension to Nomarski differential interference contrast microscopy that enables isotropic linear phase imaging. The method combines phase shifting, two directions of shear and Fourier‐space integration using a modified spiral phase transform. We simulated the method using a phantom object with spatially varying amplitude and phase. Simulated results show good agreement between the final phase image and the object phase, and demonstrate resistance to imaging noise.

Confocal differential interference contrast (DIC) microscopy: including a theoretical analysis of conventional and confocal DIC imaging

A technique for obtaining differential interference contrast (DIC) imaging using a confocal microscope system is examined and its features compared to those of existing confocal differential phase contrast (DPC) techniques as well as to conventional Nomarski DIC. A theoretical treatment of DIC imaging is presented, which takes into account the vignetting effect caused by the finite size of the lens pupils. This facilitates the making of quantitative measurements in DIC and allows the user to identify and select the most appropriate system parameters, such as the bias retardation and lateral shear of the Wollaston prism.

Three-dimensional image formation in confocal microscopy

Three‐dimensional (3‐D) imaging in confocal microscopes is considered in terms of 3‐D transfer functions. This leads to an explanation of axial imaging properties. The axial response was observed in both object‐scanning and beam‐scanning microscopes and the influence of off‐axis examination investigated. By simple processing of multi‐detector signals, imaging in both the axial and transverse directions can be improved.

Quantitative phase microscopy through differential interference imaging

An extension of Nomarski differential interference contrast microscopy enables isotropic linear phase imaging through the combination of phase shifting, two directions of shear, and Fourier space integration using a modified spiral phase transform. We apply this method to simulated and experimentally acquired images of partially absorptive test objects. A direct comparison of the computationally determined phase to the true object phase demonstrates the capabilities of the method. Simulation results predict and confirm results obtained from experimentally acquired images.

Confocal imaging of a stratified medium

The confocal imaging of stratified media (for example, thin-film structures) is investigated. A simple model is introduced for the imaging of a single layer in order to explore the axial resolution attainable. A rigorous model is also described and compared with experimental results from thin surface films. A theoretical treatment of imaging of stratified media with a continuously varying refractive index is presented, and the inverse problem of reconstructing the refractive-index profile from a confocal image is discussed.

Confocal microscopy with detector arrays

Abstract Imaging in a scanning optical microscope with a detector consisting of an array of rings is considered. It is found that both transverse and axial resolution can be increased simultaneously.

Feature extraction of chromosomes from 3-D confocal microscope images

An investigation of local energy surface detection integrated with neural network techniques for image segmentation is presented, as applied in the feature extraction of chromosomes from image datasets obtained using an experimental confocal microscope. Use of the confocal microscope enables biologists to observe dividing cells (living or preserved) within a three-dimensional (3-D) volume, that can be visualised from multiple aspects, allowing for increased structural insight. The Nomarski differential interference contrast mode used for imaging translucent specimens, such as chromosomes, produces images not suitable for volume rendering. Segmentation of the chromosomes from this data is, thus, necessary. A neural network based on competitive learning, known as Kohonens self-organizing feature map (SOFM) was used to perform segmentation, using a collection of statistics or features defining the image. Our past investigation showed that standard features such as the localized mean and variance of pixel intensities provided reasonable extraction of objects such as mitotic chromosomes, but surface detail was only moderately resolved. In this current work, a biologically inspired feature known as local energy is investigated as an alternative image statistic based on phase congruency in the image. This, along with different combinations of other image statistics, is applied in a SOFM, producing 3-D images exhibiting vast improvement in the level of detail and clearly isolating the chromosomes from the background. Index Terms-DIC, differential interference contrast, feature extraction, feature space, image segmentation, local energy, Morlet wavelet, phase congruency, self organizing feature map, SOFM.

Quantitative structured-illumination phase microscopy

We introduce a quantitative phase imaging method for homogeneous objects with a bright field transmission microscope by using an amplitude mask and a digital processing algorithm. A known amplitude pattern is imaged on the sample plane containing a thick phase object by placing an amplitude mask in the field diaphragm of the microscope. The phase object distorts the amplitude pattern according to its optical path length (OPL) profile, and the distorted pattern is recorded in a CCD detector. A digital processing algorithm then estimates the objects quantitative OPL profile based on a closed form analytical solution, which is derived using a ray optics model for objects with small OPL gradients.

The Specimen Illumination Path and Its Effect on Image Quality

The unique imaging properties of all confocal microscope systems are based on the fundamental condition that the illumination and collection (detection) optical paths contribute equally to the formation of the final image. However, many of the optical and imaging properties of confocal microscopes can be analyzed effectively by considering the illumination and detection paths to be two distinct entities. In this chapter, we explore one of these optical paths—the illumination path—and describe the ideal function and practical limitations of the total path and the optical components therein. Several other chapters in this volume present additional specific information concerning some of these optical components (e.g., light sources, intermediate optics, objective lenses). Our purpose is to treat the illumination path as an integral system and describe its contribution to the overall performance of the confocal microscope.

Colour confocal reflection microscopy using red, green and blue lasers

To obtain colour reflected confocal images we have incorporated three lasers (HeNe: 633 nm; NdYAG: 532 nm; HeCd: 442 nm) and three photomultiplier detectors into our on‐axis scanning system then adjusted the registration of the simultaneous output signals to produce full‐colour images on a video monitor. Colour confocal images were produced from multi‐stained fixed tissue as well as from natural pigments in fresh plant material. Rayleigh scattering properties of immunogold‐labelled specimens were studied to show how variations in colour response can be utilized to identify subwavelength gold particles. Colour stereo pairs were produced to illustrate the accuracy with which the three‐laser microscope system can record depth information without incurring problems due to chromatic aberration effects.